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Polymorphism of amyloid-like fibrils can be defined by the concentration of seeds.

Sneideris T, Milto K, Smirnovas V - PeerJ (2015)

Bottom Line: The strains are enciphered by different misfolded conformations.Strain-like phenomena have also been reported in a number of other amyloid-forming proteins.Our findings could explain conformational switching between amyloid strains observed in a wide variety of in vivo and in vitro experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biothermodynamics and Drug Design, Vilnius University, Institute of Biotechnology , Vilnius , Lithuania.

ABSTRACT
Prions are infectious proteins where the same protein may express distinct strains. The strains are enciphered by different misfolded conformations. Strain-like phenomena have also been reported in a number of other amyloid-forming proteins. One of the features of amyloid strains is the ability to self-propagate, maintaining a constant set of physical properties despite being propagated under conditions different from those that allowed initial formation of the strain. Here we report a cross-seeding experiment using strains formed under different conditions. Using high concentrations of seeds results in rapid elongation and new fibrils preserve the properties of the seeding fibrils. At low seed concentrations, secondary nucleation plays the major role and new fibrils gain properties predicted by the environment rather than the structure of the seeds. Our findings could explain conformational switching between amyloid strains observed in a wide variety of in vivo and in vitro experiments.

No MeSH data available.


Related in: MedlinePlus

Concentration of seeds determines the mechanism of aggregation and stability of the final strain.Different amounts of rPrP-A4M fibrils (sonicated for 300 s) were added to the solution of rMoPrP, prepared in 2 M GuHCl, 50 mM phosphate buffer, pH6. The kinetics was followed at 60 °C using Thioflavin T (ThT) fluorescence assay, five data repeats at each seed concentration plotted in (A–C). No change of ThT fluorescence was observed in samples without seeds. Denaturation profiles in GuSCN reveal different conformational stabilities of formed fibrils (D–F). Standard errors calculated from 6 measurements using Student’s t-distribution at P = 0.05.
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fig-2: Concentration of seeds determines the mechanism of aggregation and stability of the final strain.Different amounts of rPrP-A4M fibrils (sonicated for 300 s) were added to the solution of rMoPrP, prepared in 2 M GuHCl, 50 mM phosphate buffer, pH6. The kinetics was followed at 60 °C using Thioflavin T (ThT) fluorescence assay, five data repeats at each seed concentration plotted in (A–C). No change of ThT fluorescence was observed in samples without seeds. Denaturation profiles in GuSCN reveal different conformational stabilities of formed fibrils (D–F). Standard errors calculated from 6 measurements using Student’s t-distribution at P = 0.05.

Mentions: In our previous work we have described elongation kinetics at different temperatures and GuHCl concentrations, using rPrP-A2M as a seed (Milto, Michailova & Smirnovas, 2014). It was not possible to get reliable data above 2.5 M GuHCl due to depolymerization of rPrP-A2M. Thus only one way cross-seeding is possible for rPrP-A2M and rPrP-A4M strains. We followed cross-seeding kinetics using different concentrations of seeds. As seen in Fig. 2A, five percent seeds led to fast growth of amyloid-like fibrils from the very beginning, suggesting fast fibril elongation. At 1% seed volume (Fig. 2B) elongation is slower, but after some time the rate of aggregation explodes. At a lower concentration of seeds (Fig. 2C) elongation is very slow and the curve looks sigmoidal, as usually seen in case of spontaneous fibrillation; however in absence of seeds no aggregation was detected within the experimental timeframe. Fitting data suggests that the observed process can be attributed to fibril-induced secondary nucleation (see Supplemental Information). The fibril denaturation assay (Fig. 2D) revealed that stability of fibrils formed in the presence of 5% seeds (midpoint at ∼2.9 M GuSCN) is very similar to rPrP-A4M strain, which was used as a seed. At 1% seed volume (Fig. 2E), stability of fibrils is lower (midpoint at ∼2.2 M GuSCN), and at 0.2% of seeds (Fig. 2F) it is the same (midpoint at ∼1.8 M GuSCN) as the rPrP-A2M strain. This allows hypothesizing that fibrils initiated by secondary nucleation do not follow the seeding template, despite using template fibrils as nucleation sites.


Polymorphism of amyloid-like fibrils can be defined by the concentration of seeds.

Sneideris T, Milto K, Smirnovas V - PeerJ (2015)

Concentration of seeds determines the mechanism of aggregation and stability of the final strain.Different amounts of rPrP-A4M fibrils (sonicated for 300 s) were added to the solution of rMoPrP, prepared in 2 M GuHCl, 50 mM phosphate buffer, pH6. The kinetics was followed at 60 °C using Thioflavin T (ThT) fluorescence assay, five data repeats at each seed concentration plotted in (A–C). No change of ThT fluorescence was observed in samples without seeds. Denaturation profiles in GuSCN reveal different conformational stabilities of formed fibrils (D–F). Standard errors calculated from 6 measurements using Student’s t-distribution at P = 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4563235&req=5

fig-2: Concentration of seeds determines the mechanism of aggregation and stability of the final strain.Different amounts of rPrP-A4M fibrils (sonicated for 300 s) were added to the solution of rMoPrP, prepared in 2 M GuHCl, 50 mM phosphate buffer, pH6. The kinetics was followed at 60 °C using Thioflavin T (ThT) fluorescence assay, five data repeats at each seed concentration plotted in (A–C). No change of ThT fluorescence was observed in samples without seeds. Denaturation profiles in GuSCN reveal different conformational stabilities of formed fibrils (D–F). Standard errors calculated from 6 measurements using Student’s t-distribution at P = 0.05.
Mentions: In our previous work we have described elongation kinetics at different temperatures and GuHCl concentrations, using rPrP-A2M as a seed (Milto, Michailova & Smirnovas, 2014). It was not possible to get reliable data above 2.5 M GuHCl due to depolymerization of rPrP-A2M. Thus only one way cross-seeding is possible for rPrP-A2M and rPrP-A4M strains. We followed cross-seeding kinetics using different concentrations of seeds. As seen in Fig. 2A, five percent seeds led to fast growth of amyloid-like fibrils from the very beginning, suggesting fast fibril elongation. At 1% seed volume (Fig. 2B) elongation is slower, but after some time the rate of aggregation explodes. At a lower concentration of seeds (Fig. 2C) elongation is very slow and the curve looks sigmoidal, as usually seen in case of spontaneous fibrillation; however in absence of seeds no aggregation was detected within the experimental timeframe. Fitting data suggests that the observed process can be attributed to fibril-induced secondary nucleation (see Supplemental Information). The fibril denaturation assay (Fig. 2D) revealed that stability of fibrils formed in the presence of 5% seeds (midpoint at ∼2.9 M GuSCN) is very similar to rPrP-A4M strain, which was used as a seed. At 1% seed volume (Fig. 2E), stability of fibrils is lower (midpoint at ∼2.2 M GuSCN), and at 0.2% of seeds (Fig. 2F) it is the same (midpoint at ∼1.8 M GuSCN) as the rPrP-A2M strain. This allows hypothesizing that fibrils initiated by secondary nucleation do not follow the seeding template, despite using template fibrils as nucleation sites.

Bottom Line: The strains are enciphered by different misfolded conformations.Strain-like phenomena have also been reported in a number of other amyloid-forming proteins.Our findings could explain conformational switching between amyloid strains observed in a wide variety of in vivo and in vitro experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biothermodynamics and Drug Design, Vilnius University, Institute of Biotechnology , Vilnius , Lithuania.

ABSTRACT
Prions are infectious proteins where the same protein may express distinct strains. The strains are enciphered by different misfolded conformations. Strain-like phenomena have also been reported in a number of other amyloid-forming proteins. One of the features of amyloid strains is the ability to self-propagate, maintaining a constant set of physical properties despite being propagated under conditions different from those that allowed initial formation of the strain. Here we report a cross-seeding experiment using strains formed under different conditions. Using high concentrations of seeds results in rapid elongation and new fibrils preserve the properties of the seeding fibrils. At low seed concentrations, secondary nucleation plays the major role and new fibrils gain properties predicted by the environment rather than the structure of the seeds. Our findings could explain conformational switching between amyloid strains observed in a wide variety of in vivo and in vitro experiments.

No MeSH data available.


Related in: MedlinePlus